Systems, methods, and compositions for mixing and applying lyophilized biomaterials

ABSTRACT

A method of treating tissue lyophilizes a biocompatible polymer having a functionality equal to or greater than three. The method provides a protein solution. The method mixes the protein solution with the lyophilized polymer to reconstitute the polymer and form a mixture, wherein, upon mixing, the protein solution and the polymer cross-link to form a material composition. The method applies the material composition to a tissue region. The biocompatible polymer can comprise, e.g., poly(ethylene glycol) PEG. The protein solution can comprise, e.g., albumin.

RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 10/141,510, filed May 8, 2002 and entitled “Systems, Methods,and Compositions for Achieving Closure of Vascular Puncture Sites” (nowU.S. Pat. No. 7,279,001), which is a continuation-in-part of U.S. patentapplication Ser. No. 09/780,843, filed Feb. 9, 2001, and entitled“Systems, Methods, and Compositions for Achieving Closure of VascularPuncture Sites,” which is a continuation-in-part of U.S. patentapplication Ser. No. 09/283,535, filed Apr. 1, 1999, and entitled“Compositions, Systems, And Methods For Arresting or ControllingBleeding or Fluid Leakage in Body Tissue,” which is itself acontinuation-in-part of U.S. patent application Ser. No. 09/188,083,filed Nov. 6, 1998 and entitled “Compositions, Systems, and Methods forCreating in Situ, Chemically Cross-linked, Mechanical Barriers.”

FIELD OF THE INVENTION

The invention generally relates to the systems and methods fordelivering biocompatible materials to body tissue to affect desiredtherapeutic results.

BACKGROUND OF THE INVENTION

There are many therapeutic indications today that pose problems in termsof technique, cost efficiency, or efficacy, or combinations thereof.

For example, following an interventional procedure, such as angioplastyor stent placement, a 5 Fr to 9 Fr arteriotomy remains. Typically; thebleeding from the arteriotomy is controlled through pressure applied byhand, by sandbag, or by C-clamp for at least 30 minutes. While pressurewill ultimately achieve hemostasis, the excessive use and cost of healthcare personnel is incongruent with managed care goals.

Various alternative methods for sealing a vascular puncture site havebeen tried. For example, collagen plugs have been used to occlude thepuncture orifice. The collagen plugs are intended to activate plateletsand accelerate the natural healing process. Holding the collagen sealsin place using an anchor located inside the artery has also been tried.Still, patient immobilization is required until clot formationstabilizes the site. Other problems, such as distal embolization of thecollagen, rebleeding, and the need for external pressure to achievehemostasis, also persist.

As another example, devices that surgically suture the puncture sitepercutaneously have also been used. The devices require the practice offine surgical skills to place needles at a precise distance from theedges of the puncture orifice and to form an array of suture knots,which are tightened and pushed from the skin surface to the artery wallwith a knot pusher, resulting in puncture edge apposition.

There remains a need for fast and straightforward mechanical andchemical systems and methods to close vascular puncture sites and toaccelerate the patient's return to ambulatory status without pain andprolonged immobilization.

There also remains a demand for biomaterials that improve the technique,cost efficiency, and efficacy of these and other therapeuticindications.

SUMMARY OF THE INVENTION

One aspect of the invention provides a method comprising providing afirst container holding a biocompatible polymer in lyophilized form, thebiocompatible polymer having a functionality equal to or greater thanthree. The method provides a second container holding a proteinsolution. The method introduces the protein solution into the firstcontainer for mixing with the polymer in lyophilized form toreconstitute the polymer and form a mixture, wherein, upon mixing, theprotein solution and the polymer cross-link. The biocompatible polymercan comprise, e.g., poly(ethylene glycol) PEG. The protein solution cancomprise, e.g., albumin.

Another aspect of the invention provides a system comprising a firstcontainer holding a biocompatible polymer in lyophilized form, thebiocompatible polymer having a functionality equal to or greater thanthree. The system includes a second container holding a proteinsolution. The systems includes an applicator to introduce the proteinsolution into the first container for mixing with the polymer inlyophilized form to reconstitute the polymer and form a mixture,wherein, upon mixing, the protein solution and the polymer cross-link.The biocompatible polymer can comprise, e.g., poly(ethylene glycol) PEG.The protein solution can comprise, e.g., albumin.

Another aspect of the invention provides a method of treating tissue.The method lyophilizes a biocompatible polymer having a functionalityequal to or greater than three. The method provides a protein solution.The method mixes the protein solution with the lyophilized polymer toreconstitute the polymer and form a mixture, wherein, upon mixing, theprotein solution and the polymer cross-link to form a materialcomposition. The method applies the material composition to a tissueregion. The biocompatible polymer can comprise, e.g., poly(ethyleneglycol) PEG. The protein solution can comprise, e.g., albumin.

Another aspect of the method provides a method. The method lyophilizes abiocompatible polymer having a functionality equal to or greater thanthree.

The method provides a protein solution. The method mixes the proteinsolution with the lyophilized polymer to reconstitute the polymer andform a mixture, wherein, upon mixing, the protein solution and thepolymer cross-link to form a material composition. The biocompatiblepolymer can comprise, e.g., poly(ethylene glycol) PEG. The proteinsolution can comprise, e.g., albumin.

Another aspect of the invention provides a method of treating tissue.The method lyophilizes a biocompatible polymer having a functionalityequal to or greater than three. The method provides a protein solution.The method provides a catheter. The method mixes the protein solutionwith the lyophilized polymer to reconstitute the polymer and form amixture, wherein, upon mixing, the protein solution and the polymercross-link to form a material composition. The method applies thematerial composition through the catheter to a tissue region. Thebiocompatible polymer can comprise, e.g., poly(ethylene glycol) PEG. Theprotein solution can comprise, e.g., albumin.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of a system of functional instruments for closure of avascular puncture site e.g., following a vascular access procedure,comprising a vascular puncture site access assembly, to gaintranscutaneous access to the vascular puncture site for the purpose ofdelivering a biocompatible material closure composition, and a formativecomponent assembly, to house the components of the biocompatiblematerial closure composition prior to use.

FIG. 2 is an enlarged section view of the proximal end of a catheterassembly that forms a part of the vascular puncture site access assemblyshown in FIG. 1.

FIG. 3 is a cross section view of the inner and outer catheter bodiesthat comprise the catheter assembly shown in FIG. 2, taken generallyalong section line 3-3 in FIG. 2.

FIG. 4A is an enlarged section view of the distal end of a catheterassembly that forms a part of the vascular puncture site access assemblyshown in FIG. 1, showing the expandable structure carried by theassembly in a collapsed condition.

FIG. 4B is an enlarged view of the wall of the expandable structureshown in FIG. 4A, showing its open or woven configuration that allowsblood flow through the structure.

FIG. 5 is an enlarged section view of the distal end of a catheterassembly that forms a part of the vascular puncture site access assemblyshown in FIG. 1, showing the expandable structure carried by theassembly in an expanded condition.

FIG. 6 is an enlarged side section view of the junction between theexpandable structure and the outer catheter body of the catheterassembly shown in FIGS. 4A and 5.

FIGS. 7A and 7B are perspective views of alternative arrays ofcomposition delivery nozzles located on the catheter assembly that formsa part of the vascular puncture site access assembly shown in FIG. 1.

FIG. 8 is an assembled section view of the components of formativecomponent assembly shown in FIG. 1.

FIG. 9 is an enlarged view illustrating the arrangement of side holes inthe first needle of the formative component assembly shown in FIG. 1.

FIG. 10 is a perspective view of the individual components of theformative component assembly shown in FIG. 1 and further illustratingthe catheter assembly shown in FIG. 2 and the introducer/mixer assemblyshown in FIG. 1 coupled together.

FIGS. 11-16 illustrate the use of the formative component assembly todeliver a closure composition to a vascular puncture site, wherein

FIGS. 11A and 11B are perspective views illustrating the insertion ofthe vial component of the formative component assembly shown in FIG. 1into to the applicator component;

FIG. 12 is a perspective view illustrating the insertion of the syringecomponent of the formative component assembly shown in FIG. 1 into tothe applicator component;

FIG. 13 is a perspective view illustrating the procedure of coupling theassembled formative component assembly shown in FIG. 8 to theintroducer/mixer assembly, which is coupled to the catheter assembly asshown in FIG. 10;

FIG. 14 is a perspective view illustrating the advancement of thesyringe plunger component of the formative component assembly andfurther illustrating the transfer of the liquid component in the syringeinto the vial containing the solid component mixture of the liquid andthe reconstituted solid components in the vial;

FIG. 15 is a perspective view illustrating the urging of the mixturefrom the vial through the second needle component of the formativecomponent assembly and into the introducer/mixer assembly;

FIG. 16 is a perspective view illustrating the syringe and vial afterthe mixture has been transferred from the vial to the introducer/mixerassembly, and further illustrating residual mixture in the vial;

FIG. 17 is a diagrammatic view of blood vessel puncture site formed toenable the delivery of a diagnostic or therapeutic instrument through avascular sheath and over a guide wire;

FIG. 18 is a diagrammatic view of the blood vessel puncture site shownin FIG. 17, after removal of the diagnostic or therapeutic instrumentand vascular sheath, keeping the guide wire deployed;

FIG. 19 is a diagrammatic view of the blood vessel puncture site shownin FIG. 18, during deployment of the vascular puncture site accessassembly shown in FIG. 1, the access assembly being deployed over theguide wire with the expandable structure in a collapsed condition;

FIG. 20 is a diagrammatic view of the blood vessel puncture site shownin FIG. 19, with the vascular puncture site access assembly deployed andthe expandable structure in an expanded condition serving as apositioner within the blood vessel for the closure composition deliverynozzles outside the blood vessel;

FIG. 21 is a diagrammatic view of the blood vessel puncture site shownin FIG. 20, as the closure composition is being delivered through theclosure composition delivery nozzles outside the blood vessel; and

FIG. 22 is a diagrammatic view of the blood vessel puncture site shownin FIG. 21, after removal of the vascular puncture site access assemblyand after the closure composition has formed a barrier to seal thepuncture site.

DETAILED DESCRIPTION

Although the disclosure hereof is detailed and exact to enable thoseskilled in the art to practice the invention, the physical embodimentsherein disclosed merely exemplify the invention that may be embodied inother specific structure. While the preferred embodiment has beendescribed, the details may be changed without departing from theinvention, which is defined by the claims.

The systems and methods disclosed herein are shown in the particularcontext of closing a vascular puncture site. That is because the systemsand methods are well suited for use in this indication, and thisindication thus provides a representative embodiment for purposes ofdescription. Still, it should be appreciated that the systems andmethods described can, with appropriate modification (if necessary), beused for diverse other indications as well, and in conjunction withdelivery mechanisms that are not necessarily catheter-based. Forexample, the systems and methods can be used with delivery mechanismswhich spray materials, e.g., for the purpose of tissue sealing oradhesion prevention. As another example, the systems and methods can beused with delivery mechanisms which use cannulas, e.g., for the purposeof filling tissue voids or aneurysms, or for tissue augmentation. As yetanother example, the systems and methods can be used to deliver drug orcells to targeted locations.

System Overview

FIG. 1 shows a system 10 of functional instruments for closure of avascular puncture site e.g., following a vascular access procedure.

As will be described in greater detail, the instruments of the system 10are, during use, deployed in a purposeful manner to gain transcutaneousaccess to a vascular puncture site. The instruments of the system 10 aremanipulated to place a biocompatible material composition outside theblood vessel at the puncture site. The biocompatible materialcomposition produces a solid, three dimensional matrix that closes thepuncture site.

In a preferred embodiment, the biocompatible material composition iscomprised of two or more formative components which are mixed in aliquid state while being delivered by the system 10 transcutaneously tothe puncture site. Upon mixing, the formative components react, in aprocess called “gelation,” to transform in situ from the liquid state,to a semi-solid (gel) state, and then to the biocompatible solid state.

In the solid state, the composition takes the form of a non-liquid,three-dimensional network. Desirably, the solid material compositionexhibits adhesive strength (adhering it to adjacent tissue), cohesivestrength (forming a mechanical barrier that is resistant to bloodpressure and blood seepage), and elasticity (accommodating the normalstresses and strains of everyday activity). These properties provide aneffective closure to the vascular puncture site.

The solid material composition is also capable of transforming over timeby physiological mechanisms from the solid state to a biocompatibleliquid state, which can be cleared by the body, in a process called“degradation.”

As FIG. 1 shows, in one embodiment, the system 10 can be contained,prior to use, in two functional kits 12 and 14.

The first kit 12 contains a vascular puncture site access assembly 16.The purpose of the access assembly 16 is to gain transcutaneous accessto the vascular puncture site for the purpose of delivering thebiocompatible material composition.

The second kit 14 contains a formative component assembly 18 anddirections for use 19. The purpose of the formative component assembly18 is to house the components of the biocompatible material compositionprior to use. As will be described in greater detail later, thesecomponents are mixed and delivered by the access assembly 16 to thepuncture site. The directions for use 19 provide the user with astep-by-step procedure and information for use of the assembly 18, aswill be described in greater detail later (see FIGS. 11-16).

The kits 12 and 14 can take various forms. In the illustratedembodiment, each kit 12 and 14 comprises a sterile (e.g, sterilized byethylene oxide gas), wrapped assembly.

The Access Assembly

As FIG. 1 shows, the access assembly 16 comprises a catheter assembly 20and a component introducer/mixer assembly 22.

The Catheter Assembly

The catheter assembly comprises a flexible inner catheter body 24 thatis slidably carried within a flexible outer catheter body 24 (see FIGS.2 to 5). The inner and outer catheter bodies 24 and 26 can be made froman extruded plastic material, e.g., PEBAX™ material. The outsidediameter of the outer catheter body 26 can vary, e.g., from 6 Fr. to 10Fr.

The outside diameter of the outer catheter body 26 is sized to seal thetissue track 34 through which it is introduced, so that its presence ishemostatic (see FIGS. 19-21). The tissue track 34 typically will havebeen previously formed by a vascular introducer or cannula 28 (see FIG.17), through which the desired therapeutic or diagnostic instrument isfirst introduced (typically over a guide wire 32) through a puncturesite 36 into the vessel, e.g., to perform coronary angioplasty. Afterperforming the intended procedure, the instrument 30 and introducer 28are withdrawn (see FIG. 18), leaving the puncture site 36 and the tissuetrack 34. The outside diameter of the outer catheter body 26 is selectedto match the outside diameter of the vascular introducer 28, so that theouter catheter body 26, when deployed, will block substantial flow ofblood from the puncture site 36 up the tissue track 34.

The proximal end of the outer catheter body 26 is secured, e.g., byadhesive, to the distal end of a preformed y-shaped adapter 38 (see FIG.2). The adapter 38 serves as a handle for the entire catheter assembly20. A strain relief sheath 40 desirably encompasses the outer catheterbody 26 adjacent the handle 38.

The proximal end of the inner catheter body 24 extends through andbeyond the handle 38. The exposed end of the inner catheter body 24desirably carries a luer fitting 42, so that a flushing fluid can beintroduced through the inner catheter body 24. The inside diameter ofthe inner catheter body 24 defines an interior lumen 44 (see FIG. 3)that is sized to accommodate passage of the guide wire 32.

A carrier 46 is carried on a track 48 in the handle 38 for fore and aftsliding movement. The inner catheter body 24 is adhesively securedwithin the sliding carrier 46, so that fore and aft movement of thecarrier 46 in the track 48 affects sliding movement of the innercatheter body 24 (as FIGS. 4A and 5 show). In response to forwardmovement of the carrier 46 (as FIG. 4A shows), the inner catheter body24 slides in a distal direction within the outer catheter body 26. Inresponse to aft movement of the carrier 46 (as FIG. 5 shows), the innercatheter body 24 slides in a proximal direction within the outercatheter body 26.

A spring biased latch mechanism 50 is desirably coupled to the carrier46. The latch mechanism 50 snap-fits into detents 52 (shown in FIG. 2)at the proximal and distal ends of the track 48, to releasably lock thecarrier 46 against movement. Finger pressure releases the spring biasedlatch mechanism 50 from the detents 52, to release the carrier 46 formovement between the proximal and distal detents 52.

The interior diameter of the outer catheter body 26 (see FIGS. 3 to 5)is larger than the exterior diameter of the inner catheter body 24. Ininterior passage 54 is thereby defined between them (see FIG. 3). A port56 on the handle 38 communicates with the passage 54 (see FIG. 2). Theport 56 terminates with the component introducer/mixer assembly 22through intermediate tubing 58. Liquid components introduced through theassembly 22 exit the passage 54 through one or more nozzles 60 formednear the distal end of the outer catheter body 26 (see FIG. 6). As FIGS.3 and 6 show, a thin wall tube 62 (extruded, e.g., from a polyimidematerial) desirably covers the inner catheter body 24, to prevent liquidcomponents within the passage 54 from adhesively bonding the innercatheter body 24 to the outer catheter body 26. Free sliding motion ofthe inner catheter body 24 within the tube 62 is thereby preserved.

The nozzles 60 can be arranged in different delivery patterns. In oneembodiment (as FIG. 7A shows), an array of nozzles 60, circumferentiallyspaced apart, is provided. In another embodiment (as FIG. 7B shows), thenozzles 60A and 60B are spaced apart along the axis of the outercatheter body 26, as well as being staggered to face differentdirections about the axis.

The diameter of the nozzles 60 can also vary (e.g., from 0.02″ to0.035″). The nozzles 60 can all share the same diameter. Alternatively,the nozzles 60 can have different diameters, to create preferential flowpatterns (the liquid composition following the path of less flowresistance in preference to a path of greater flow resistance).

It is desired that the nozzles 60 reside outside the blood vessel whenthe material composition is introduced. To help locate the nozzles 60outside the blood vessel, the catheter assembly 20 includes anexpandable structure 64 located near to and distally of the nozzles 60(see FIGS. 4A and 5).

The wall 66 of the structure 64 desirably comprises an open or woven orbraided structure comprising interlaced or intersecting strands orthreads 68 (see FIG. 4B), e.g., made from an inert biocompatablepolymeric material, such as nylon. Alternatively, the outer catheterbody 26 can itself be slotted at circumferentially spaced locations toform the structure 64. The proximal end of the structure 64 is secured(see FIG. 6), e.g., by a fuse joint 70, about a gland member 72 thatencircles the thin wall tube 62. As FIG. 6 also shows, the distal end ofthe outer catheter body 26 is also secured, e.g., by adhesive, to thegland member 72. An o-ring 74 is also desired placed within the glandmember 72 to prevent leakage of liquid components from the passage 54into the interior of the structure 64.

The distal end of the structure 64 is secured, e.g., by adhesive or ashrink-fit sleeve, to a region of the inner catheter body 24 thatextends beyond the outer catheter body 26. The inner catheter body 24also extends a distance distally beyond the structure 64, forming aleader 76 (see FIGS. 4A and 5). In use, the leader 76 is located insidethe blood vessel immediately interior to the puncture site 36 (like theleader 76 in FIGS. 19 and 20). In use, the array of nozzles 60 islocated outside the blood vessel exterior to the puncture site 36 (likethe nozzles 60 in FIG. 20). Sliding movement of the inner catheter body24 relative to the outer catheter body 26 serves to mechanically expand(see FIG. 5) and collapse (see FIG. 4A) the structure 64, so that thisdesired positioning of the nozzles 60 and leader 76 can be achieved.

Since, in the illustrated embodiment, the structure 64 possesses a wallthat is open or woven, the structure 64 permits blood flow through it,thereby presenting a minimal disruption of blood flow in the vesselduring use. Due to the open or woven configuration of the structure 64,the positioner can be deployed in an expanded state within the arteryprior to being seated against the interior of the vessel wall, withminimal disruption of blood flow. This allows the physician to proceedwith the deployment and positioning of the structure 64 within thevessel in a deliberate fashion, without being rushed due to ancillaryconsiderations of attendant blood flow disruption. The open structure 64can be deployed while a patient is in an operating room, and leftdeployed while the patient is wheeled from the operating room to anothersuite, where the vessel closure procedure is completed. In this way, theoperating room, its staff, and its equipment are made available foranother procedure while the vessel closure procedure is completed inanother setting by a medically trained person, who need not be a medicaldoctor.

Desirably, radiopaque marker bands 78 are secured to the proximal anddistal ends of the structure 64, as well as to the distal-most end ofthe leader 76. Preferably, the three markers 78 appear at equidistantintervals when the structure 64 is in its collapsed or stowed condition.Thus, when the structure 64 is in its expanded condition, the markers 78no longer appear equidistant. In this way, the physician can readilygauge by fluoroscopy the location of the distal-most end of the innercatheter body 24, as well as the distance between the ends of thestructure 64 and, thereby, assess the position and configuration of theinner catheter body 24 and the structure 64 near the puncture site 36.

The Component Introducer/Mixer Assembly

Before mixing, the components for the material composition are housed inthe formative component assembly 18 contained in the kit 14 (see FIG.1), which will be described in greater detail later.

As FIG. 10 shows, the proximal end of the introducer/mixer assembly 22includes a length of flexible intermediate tubing 58 that couples to theport 56 of the y-adapter/handle 38. The distal end of the assembly 22includes a luer fitting 84 that couples to the formative componentassembly 18 (see also FIG. 13).

Communicating with the tubing 58 in the direction of flow into thepassage 54, are an in-line syringe activated check valve 86, an in-linemixer 88, and an in-line air accumulator 90.

The in-line syringe activated check valve 86 can take various forms. Inthe illustrated embodiment, the valve 84 takes the form of aconventional, needleless slip luer lock valve made by Qosina (Edgewood,N.Y.), Product Number 80360. The valve 84 is normally closed to preventback flow of blood or other liquid material through the tubing 58. Backflow of blood, in particular, from the passage is undesirable, as itcreates the potential for blood contact and deposits material in theintroducer/mixer assembly 22 that can interfere or compete with thedesired reaction between the liquid components that form the materialcomposition. Connection of a conventional luer fitting carried by theformative component assembly 18 (for example, fitting 132 shown in FIGS.10 and 13) opens the valve 86 to allow the introduction of the liquidcomponents that form the material composition.

The components of the material composition come into contact in theliquid state in the in-line mixer 88. In this way, effective mixing canbe achieved outside the catheter assembly 20 that is not dependentsolely upon the dimensions or lengths of the flow paths within thecatheter assembly 20. The mixer 88 comprises a mixing structure, whichcan vary. For example, the mixer 88 can comprise a spiral mixermanufactured by TAH Industries, Inc. (Robbinsville, N.J.), Part Number121-090-08.

The in-line air accumulator 90 comprises a chamber that has an interiorvolume sized to trap air that can reside in the material compositionapplicator at time of use.

The Formative Component Assembly

The components forming the material composition can vary. Generallyspeaking, however, the components will include a solid component and aliquid component, which serves as a diluent for the solid component.Mixing of these two components initiates a chemical reaction, by whichthe liquid mixture transforms into a solid composition. It is thepurpose of the formative component assembly 18 to facilitate the mixingof these two components and introduction of the mixture into theintroducer/mixer assembly 22 and delivery to the catheter assembly 20.

The formative component assembly 18 can comprise individual syringes inwhich the components are separately contained. Further details of thisarrangement are disclosed in copending U.S. patent application Ser. No.09/187,384, filed Nov. 6, 1998 and entitled “Systems and Methods forApplying Cross-Linked Mechanical Barriers,” which is incorporated hereinby reference.

With reference to FIG. 8, an alternative arrangement provides a unitaryapplicator 92 in which a vial 94 holding a solid component 96 and asyringe 98 holding a liquid component 100 can be placed and keptseparate in interior compartments.

Axial advancement of the syringe plunger 102 propels the liquid 100 intothe vial 94 and brings the two components 96 and 100 together within thevial 94 by placing the solid component 96 into suspension within theliquid component 100. The force created by this process also urges theliquid suspension into the introducer/mixer assembly 22 for furthermixing and delivery to the catheter assembly 20.

The applicator 92 includes a partition 104 that divides the applicator92 into a first compartment 106 and a second compartment 108, eachhaving an open end 110. The first compartment 106 is sized andconfigured to receive and hold the vial 94. The first compartment 106includes a flanged end region 112 that serves to support the applicator92 in an upright position (e.g., standing on a table). The flangedregion 112 further serves to receive a cap 114, as will be described ingreater detail later. The second compartment 108 is sized and configuredto receive and hold the syringe 98. While the illustrated embodimentshows the applicator 92 and compartments 106 and 108 having a generallycylindrical shape, the invention contemplates other configurations notnecessarily accommodating a vial 94 and/or syringe 98.

The applicator 92 can be made of any suitable inert, rigid plastic ormetal material. In a representative embodiment, the first compartment106 is 2½% inches long, the second compartment 108 is 2 inches long, andthe applicator 92 is 1 inch high. This arrangement readily accommodatesa conventional vial 94 and a conventional syringe 98.

The syringe 98 can be a conventional syringe 98 having a plunger 102.The dispensing end 116 includes a luer fitting 118. The syringe 98 isaseptically pre-filled with the liquid component 100 and a cap 119 isplaced over the dispensing end 116 to prevent leakage and evaporation ofthe contents.

A first needle 120 extends along the central line axis of the applicator92 and couples the syringe 98 to the vial 94 via a luer fitting 122 thatmates with the luer fitting 118 on the syringe 98. The needle 120thereby provides communication between the first and second compartments106 and 108. Desirably, the needle 120 includes a plurality of sideholes 124 that serve to uniformly introduce the contents of the syringe98 into the vial 94 (see FIG. 9).

A second needle 126 is offset from the central line axis of theapplicator 92 and serves to couple the vial 94 to a molded passage 128that traverses the wall of the second compartment 108. The moldedpassage 128 is coupled to the proximal end of a length of flexibletubing 130. The distal end of the tubing 130 includes a luer fitting 132adapted to couple to the leur fitting 84 on the introducer/mixerassembly 22. This arrangement provides fluid communication between thevial 94 and the introducer/mixer assembly 22. Optionally, an in-line airvent 131 (shown in phantom lines in FIG. 10), made, e.g., from asintered plastic material, can be located in the tubing 130, orotherwise placed in communication with the tubing 130, to allow residualair to vent from fluid prior to entering the introducer/mixer assembly22.

The vial 94 is a conventional pharmaceutical vial 94 sized to hold thesolid component 96 and a pre-defined volume of the liquid component 100,i.e., the volume of liquid component 100 pre-filled in the syringe 98.The vial 94 includes a septum 134 configured to be pierced andpenetrated by the needles 120 and 126 when the vial 94 is properlypositioned within the first compartment 106.

To aid in positioning and securing of the vial 94 within the compartment106, the applicator 92 includes a selectively removable cap 114, aspreviously noted. The cap 114 mates with the applicator 92, e.g., bysnap-fit engagement with the flanged region 112 on the applicator 92.Desirably, the cap 114 extends into the first compartment 106 toposition and hold the vial 94 in a desired position after the septum 134has been pierced by the needles 120 and 126.

FIG. 10 shows the individual components of the formative componentassembly 18. In use, the physician (or assistant) removes the cap 114from the applicator 92. As seen in FIG. 11A, with the cap 114 removed,the physician slides the first compartment 106 over the vial 94. Duringthis step, the vial 94 can be placed on a counter, table, or other flatsupport surface. As seen in FIG. 11B, the cap 114 is then placed beneaththe vial 94 (e.g., on the counter or table), and the physician continuesto slide the first compartment 106 over the vial 94, to finish piercingthe vial septum 134 with the needles 120 and 126 and locating the vial94 fully into the first compartment 106. The cap 114 thereafter holdsthe vial 94 in this position.

The cap 119 is then removed from the syringe 98 and residual air isexpressed from the syringe 98, e.g., by holding the syringe 98 with thedispensing end 116 upright and gently tapping the syringe 98 untilessentially all of the residual air rises to the dispensing end 116 andthen advancing the plunger 102 until the air is expelled (not shown).

As FIG. 12 shows, the syringe 98 is then placed within the secondcompartment 108 and rotated (represented by arrow) to couple the syringe98 to the first needle 120 through leur fittings 118 and 122. With thesyringe 98 and vial 94 in place within the applicator 92 and theformative assembly 18 ready for use, as seen in FIG. 13, the assembly 18can then be coupled to the introducer/mixer assembly 22 (shown inphantom lines) by coupling (represented by arrows) leur fittings 84 and132.

As will be apparent, alternatively, the syringe 98 can be coupled to thefirst needle 120 prior to the vial 94 being placed in the firstcompartment 106.

With reference now to FIG. 14, the formative component assembly 18 isthen placed in an upright position (i.e., vial septum 134 pointingupward and dispensing end 116 of the syringe 98 pointing downward). Theplunger 102 is then advanced (represented by arrow) to transfer thecontents of the syringe 98 through the first needle 120 into the vial94. If desired, the assembly 18 can be stood on a counter, table, orother flat surface as the plunger 102 is advanced. Alternatively, theplunger 102 can be advanced in conventional fashion by the thumb of thephysician while the syringe 98, with attached applicator 92, are heldbetween the forefinger and middle finger, as FIG. 14 shows.

The propulsion of the liquid component 100 into the vial 94reconstitutes the solid component 96, mixes the components 96 and 100(represented by arrows in FIG. 14), and begins the reaction process. Aspreviously noted, side holes 124 in the first needle 120 assurecomponents 96 and 100 mix quickly and uniformly (see FIG. 9).

Fluid pressure created by operation of the syringe 98 urges the mixtureinto and through the second needle 126, into the introducer/mixerassembly 22, as indicated by arrows in FIG. 15. The introducer/mixerassembly 22 further mixes the mixture and rids the fluid path ofresidual air, as previously described. The mixture flows through theintroducer/mixer assembly 22 and through the catheter assembly 20 andexits the assembly 22 through the nozzles 60, as also previouslydescribed.

With reference now to FIG. 16, the plunger 102 is advanced untilessentially all of the liquid component 100 is transferred from thesyringe 98 to the vial 94. Generally concurrently, the mixture istransferred from the vial 94 into the introducer/mixer 22, with onlyminimal residual mixture remaining in the vial 94. As will apparent toone skilled in the art, the volume of components 96 and 100 arecalculated to account for this residual volume.

It should be appreciated that the applicator 92 can be coupled todiverse forms of fluid delivery systems. It can, as shown, be coupled toa catheter-based system. It can alternatively, depending upon theindicated use, be coupled to a spray applicator or to a cannula.

The Material Composition

The components 96 and 100 of the material composition can vary. In apreferred embodiment, the solid component 96 comprises an electrophilic(electrode withdrawing) material having a functionality of at leastthree. The liquid component 100 comprises a solution containing anucleophilic (electron donator) material and a buffer. When mixed underproper reaction conditions, the electrophilic material and bufferednucleophilic material react, by cross-linking with each other. Thecross-linking of the components form the composition. The compositionphysically forms a mechanical barrier 136 (see FIG. 22), which can alsobe characterized as a hydrogel.

The type and concentration of the buffer material controls the pH of theliquid and solid components 100 and 96, when brought into contact formixing. The buffer material desirably establishes an initial pH innumeric terms, as well regulates change of the pH over time (acharacteristic that will be called the “buffering capacity”).

The barrier composition 136 exhibits desired mechanical properties.These properties include adhesive strength (adhering it to adjacenttissue), cohesive strength (forming a mechanical barrier that isresistant to blood pressure and blood seepage), and elasticity(accommodating the normal stresses and strains of everyday activity).These properties, as well as the relative rapid rate of gelation thatcan be achieved, serve to provide a fast and effective closure to thevascular puncture site.

The barrier composition 136 is also capable of transforming over time byphysiological mechanisms from the solid state to a biocompatible liquidstate, which can be cleared by the body, in a process called“degradation.”

The time period that begins when the electrophilic, nucleophilic, andbuffer components have been mixed and ends when the composition hasreached the semi-solid (gel) state will be called the “gelation time.”When in this state, the barrier composition 136 possesses sufficientcohesive and adhesive strength to impede blood flow, but still retains aself-sealing property, possessing the capacity to close in upon and sealthe tract left by the catheter in the composition when the physicianremoves the catheter. For sealing a vascular puncture site, the barriercomposition 136 preferably possesses a gelation time that is in therange of fifteen to sixty seconds. A gelation time in the range offifteen to thirty seconds is most preferred. This period allows thecomponents forming the barrier composition 136 to flow first in a liquidstate, and then in the semi-solid (gel) state, outward along the axis ofthe blood vessel. The flow of components during gelation fills surfaceirregularities in the tissue region of the vascular puncture site,before solidification occurs. A gelation time period of between 10 and40 seconds also falls well within the time period a physician typicallyneeds to manipulate and remove the catheter assembly 20 after deliveryof the components to the puncture site 36. With an experiencedphysician, the catheter manipulation and removal time period can be asquick as 10 to 40 seconds, but it can extend, due to circumstances,upwards to 2 minutes. With a gelation time falling within the preferredrange, the formation of the barrier composition does not require aphysician to “watch the clock,” but rather attend only to the normaltasks of injecting the material and then manipulating and removing thecatheter assembly. With a gelation time falling within the preferredrange, it has been discovered that, if the catheter assembly 20 isremoved in 15 seconds to 2 minutes following initial mixing, the barriercomposition 136 has reached a physical state capable of performing itsintended function, while still accommodating a sealed withdrawal of thecatheter assembly 20. Desirably, after removal of the catheter assembly20, the physician applies localized and temporary finger pressure to theskin surface above the barrier composition 136 for a period of about 5minutes, to aid in the closure of the catheter tract in the composition,as the composition 136 reaches its solid state.

The barrier composition 136 preferably possesses sufficient adhesivestrength to prevent dislodging from the arteriotomy, once formed. Thecomposition 136 also has sufficient cohesive strength to prevent ruptureunder arterial pressure, i.e., up to about 200 mm Hg. The barriercomposition 136 seals the arteriotomy for up to 15 days post-applicationbefore loss of mechanical properties through degradation, and degradesby 30 to 90 days post-application.

The gelation time (which indicates the rate at which the cross-linkingreaction occurs) is controlled, inter alia, by the reaction pH, whichthe buffer component establishes. The reaction pH controls thereactivity of nucleophilic groups in the second component 100, whichreact with the electrophilic groups in the first component 96. Generallyspeaking, the higher the reaction pH is, the larger is the fraction ofnucleophilic groups available for reaction with the electrophilicgroups, and vice versa.

To achieve a relatively rapid gelation time, a relatively high initialreaction pH (which, for the illustrated components, is above 8) isdesirable at the time initial mixing of the components occurs. On theother hand, by the time the mixture is brought into contact with bodytissue at the vascular puncture site, it is desirable that the mixturepossess a more physiologically tolerated pH level (approximately 7.4).

However, it has been discovered that, if the initial reaction pH is toohigh (which, for the illustrated components, is believed to be a pHapproaching about 9), the gelation time may be too rapid to consistentlyaccommodate the time period a physician typically requires to remove thecatheter, particularly if the time period approaches the two minutemark. In this instance, by the two minute mark, substantialsolidification of the composition 136 can occur, and the composition 136can lack the cross-linking capacity to close in about the catheter tractleft in the composition upon removal of the catheter. Under thesecircumstances, blood leakage and hematoma formation can result afterremoval of the catheter assembly 20.

Achieving and sustaining a reaction pH to meet a targeted gelation timeis therefore a critical criteria. It has been discovered that, bypurposeful selection of the electrophilic, nucleophilic, and buffercomponents, (i) an initially high reaction pH can be established that isconducive to rapid gelation, before contact with body tissue occurs, and(ii) the reaction pH can be lowered as gelation progresses, as themixture is delivered through the catheter into contact with body tissueat the vascular puncture site 36. At the same time, by purposefulselection of the components, the rate at which the pH is lowered duringdelivery can be mediated, so that gelation is sustained at a rate thatmeets the gelation time requirements to achieve the desired in situformation of the composition 136, one that also possesses sufficientcross-linking capacity to close about the catheter tract followingremoval of the catheter assembly 20 after a time period a physiciantypically needs to perform this task.

The Electrophilic Component

In its most preferred form, the electrophilic (electrode withdrawing)material 96 comprises a hydrophilic, biocompatible polymer that iselectrophilically derivatized with a functionality of at least three.Examples include poly(ethylene glycol), poly(ethylene oxide), poly(vinylalcohol), poly(vinylpyrrolidinone), poly(ethyloxazoline), andpoly(ethylene glycol)-co-poly(propylene glycol) block copolymers.

As used herein, a polymer meeting the above criteria is one that beginswith a multiple arm core (e.g., pentaerythritol) and not a bifunctionalstarting material, and which is synthesized to a desired molecularweight (by derivatizing the end groups), such that polymers withfunctional groups greater than or equal to three constitute (accordingto gel permeation chromotography—GPC) at least 50% or more of thepolymer blend.

The material 96 is not restricted to synthetic polymers, aspolysaccharides, carbohydrates, and proteins could be electrophilicallyderivatized with a functionality of at least three. In addition, hybridproteins with one or more substitutions, deletions, or additions in theprimary structure may be used as the material 96. In this arrangement,the protein's primary structure is not restricted to those found innature, as an amino acid sequence can be synthetically designed toachieve a particular structure and/or function and then incorporatedinto the material. The protein of the polymer material 96 can berecombinantly produced or collected from naturally occurring sources.

Preferably, the polymer material 96 is comprised of poly(ethyleneglycol) (PEG) with a molecular weight preferably between 9,000 and12,000, and most preferably 10,500±1500. PEG has been demonstrated to bebiocompatible and non-toxic in a variety of physiological applications.The preferred concentrations of the polymer are 5% to 35% w/w, morepreferably 5% to 20% w/w. The polymer can be dissolved in a variety ofsolutions, but sterile water is preferred.

The most preferred polymer material 96 can be generally expressed ascompounds of the formula:PEG-(DCR-CG)_(n)

Where:

-   -   DCR is a degradation control region.    -   CG is a cross-linking group.    -   n≧3

The electrophilic CG is responsible for the cross-linking of thepreferred nucleophilic material 96, as well as binding the composition136 to the like material in the surrounding tissue, as will be describedlater. The CG can be selected to selectively react with thiols,selectively react with amines, or react with thiols and amines. CG'sthat are selective to thiols include vinyl sulfone, N-ethyl maleimide,iodoacetamide, and orthopyridyl disulfide. CG's that are selective toamines include aldehydes. Non-selective electrophilic groups includeactive esters, epoxides, oxycarbonylimidazole, nitrophenyl carbonates,tresylate, mesylate, tosylate, and isocyanate. The preferred CG's areactive esters, more preferred, an ester of N-hydroxysuccinimide. Theactive esters are preferred since they react rapidly with nucleophilicgroups and have a non-toxic leaving group, e.g., hydroxysuccinimide.

The concentration of the CG in the polymer material 96 can be used tocontrol the rate of gelation. However, changes in this concentrationtypically also result in changes in the desired mechanical properties ofthe hydrogel.

The rate of degradation is controlled by the degradation control region(DCR), the concentration of the CG's in the polymer solution, and theconcentration of the nucleophilic groups in the protein solution.Changes in these concentrations also typically result in changes in themechanical properties of the hydrogel, as well as the rate ofdegradation.

The rate of degradation (which desirably occurs in about 30 days) isbest controlled by the selection of the chemical moiety in thedegradation control region, DCR. If degradation is not desired, a DCRcan be selected to prevent biodegradation or the material can be createdwithout a DCR. However, if degradation is desired, a hydrolytically orenzymatically degradable DCR can be selected. Examples of hydrolyticallydegradable moieties include saturated di-acids, unsaturated di-acids,poly(glycolic acid), poly(DL-lactic acid), poly(L-lactic acid),poly(ξ-caprolactone), poly(δ-valero-lactone), poly(γ-butyrolactone),poly(amino acids), poly(anhydrides), poly(orthoesters),poly(ortho-carbonates), and poly(phosphoesters), and derivativesthereof. A preferred hydrolytically degradable DCR is gluturate.Examples of enzymatically degradable DCR's include Leu-Gly-Pro-Ala(collagenase sensitive linkage) and Gly-Pro-Lys (plasmin sensitivelinkage). It should also be appreciated that the DCR could containcombinations of degradable groups, e.g. poly(glycolic acid) and di-acid.

While the preferred polymer is a multi-armed structure, a linear polymerwith a functionality, or reactive groups per molecule, of at least threecan also be used. The utility of a given PEG polymer significantlyincreases when the functionality is increased to be greater than orequal to three. The observed incremental increase in functionalityoccurs when the functionality is increased from two to three, and againwhen the functionality is increased from three to four. Furtherincremental increases are minimal when the functionality exceeds aboutfour.

A preferred polymer may be purchased from SunBio Company ((PEG-SG)₄,having a molecular weight of 10,500±1500)(which will sometimes be calledthe “SunBio PEG”).

The Nucleophilic Component

In a most preferred embodiment, the nucleophilic material 100 includesnon-immunogenic, hydrophilic proteins. Examples include serum, serumfractions, and solutions of albumin, gelatin, antibodies, fibrinogen,and serum proteins. In addition, water soluble derivatives ofhydrophobic proteins can be used. Examples include solutions ofcollagen, elastin, chitosan, and hyaluronic acid. In addition, hybridproteins with one or more substitutions, deletions, or additions in theprimary structure may be used.

Furthermore, the primary protein structure need not be restricted tothose found in nature. An amino acid sequence can be syntheticallydesigned to achieve a particular structure and/or function and thenincorporated into the nucleophilic material 100. The protein can berecombinantly produced or collected from naturally occurring sources.

The preferred protein solution is 25% human serum albumin, USP. Humanserum albumin is preferred due to its biocompatibility and its readyavailability.

The uses of PEG polymers with functionality of greater than threeprovides a surprising advantage when albumin is used as the nucleophilicmaterial 100. When cross-linked with higher functionality PEG polymers,the concentration of albumin can be reduced to 25% and below. Past usesof difunctional PEG polymers require concentrations of albumin wellabove 25%, e.g. 35% to 45%. Use of lower concentrations of albuminresult in superior tissue sealing properties with increased elasticity,a further desired result. Additionally, 25% human serum albumin, USP iscommercially available from several sources, however higherconcentrations of human serum albumin, USP are not commerciallyavailable. By using commercially available materials, the dialysis andultrafiltration of the albumin solution, as disclosed in the prior art,is eliminated, significantly reducing the cost and complexity of thepreparation of the albumin solution.

To minimize the liberation of heat during the cross-linking reaction,the concentration of the cross-linking groups of the fundamental polymercomponent is preferably kept less than 5% of the total mass of thereactive solution, and more preferably about 1% or less. The lowconcentration of the cross-linking group is also beneficial so that theamount of the leaving group is also minimized. In a typical clinicalapplication, about 50 mg of a non-toxic leaving group is produced duringthe cross-linking reaction, a further desired result. In a preferredembodiment, the CG comprising an N-hydroxysuccinimide ester hasdemonstrated ability to participate in the cross-linking reaction withalbumin without eliciting adverse immune responses in humans.

The Buffer Component

In the most preferred embodiment, a PEG reactive ester reacts with theamino groups of the albumin and other tissue proteins, with the releaseof N-hydroxysuccinimide and the formation of a link between the PEG andthe protein. When there are multiple reactive ester groups per PEGmolecule, and each protein has many reactive groups, a network of linksform, binding all the albumin molecules to each other and to adjacenttissue proteins.

This reaction with protein amino groups is not the only reaction thatthe PEG reactive ester can undergo. It can also react with water (i.e.,hydrolyze), thereby losing its ability to react with protein. For thisreason, the PEG reactive ester must be stored dry before use anddissolved under conditions where it does not hydrolyze rapidly. Thestorage container for the PEG material desirably is evacuated by use ofa vacuum, and the PEG material is stored therein under an inert gas,such as Argon or Nitrogen. Another method of packaging the PEG materialis to lyophilize the PEG material and store it under vacuum, or under aninert gas, such as Argon or Nitrogen, as will be described in greaterdetail later. Lyophilization provides the benefits of long term storageand product stability, as well as allows rapid dissolution of the PEGmaterial in water.

The conditions that speed up hydrolysis tend to parallel those thatspeed up the reaction with protein; namely, increased temperature;increased concentration; and increased pH (i.e., increased alkali). Inthe illustrated embodiment, temperature cannot be easily varied, sovarying the concentrations and the pH are the primary methods ofcontrol.

It is the purpose of the buffer material (which is added to thenucleophilic albumin material 100 prior to mixing with the electrophilicPEG material 96) to establish an initial pH to achieve a desiredgelation time, and to sustain the pH as added acid is produced by therelease of N-hydroxysuccinimide during cross linking and hydrolysis.

pH is the special scale of measurement established to define theconcentration in water of acid (H+) or alkali (OH—) (which, strictlyspeaking, indicates hydrogen ion activity). In the pH scale, solutionsof acid (H+) in water have a low pH, neutrality is around pH 7, andsolutions of base (OH—) in water have a high pH. The pH scale islogarithmic. A change of one pH unit (e.g., from pH 9 to pH 10)corresponds to a ten-fold change in concentration (i.e., hydrogen ionactivity). Thus, reactions which are increased by alkali, such ashydrolysis of PEG reactive ester, are expected to increase in rate by afactor of ten for each unit increase in pH.

The buffer material is a mixture of molecules, added to the albumin,that can moderate pH changes by reacting reversibly with added acid (H+)or base (OH—). The pH moderating effect can be measured by titration,i.e., by adding increasing amounts of H+ or OH— to the buffer material,measuring the pH at each step, and comparing the pH changes to that of asimilar solution without the buffer.

Different buffers exert a maximum pH moderating effect (i.e., the leastchange in pH with added H+ or OH—) at different pH's. The pH at which agiven buffer exerts its maximum pH moderating effect is called its pK.

Even when the pH matches the pK for a given buffer, added acid or basewill produce some change in pH. As the pH changes from the pK value, themoderating effect of the buffer decreases progressively (e.g., 67% lessat +/−1 pH unit from pK, and 90% less at +/−1.6 pH unit from pK). Themoderating effect is also proportional to the buffer concentration.Thus, increasing the buffer concentration increases the ability tomoderate pH changes.

The overall buffering effect at any pH is the sum of all bufferingspecies present, and has also been earlier called the bufferingcapacity. The higher the buffering capacity, the more acid or base mustbe added to produce a given pH change. Stated differently, the higherthe buffering capacity, the longer a given buffer is able to sustain atargeted pH range as acid or base is being added to change the pH.

Albumin itself contains amino, carboxyl, and other groups, which canreversible react with acid and base. That is, albumin itself is abuffer. Also, due to the many different buffering groups that albuminpossesses, albumin (e.g., Plasbumin) can buffer over a relatively broadpH range, from below pH 6 to over pH 10. However, it has been discoveredthat albumin lacks the buffering capacity to, by itself, counterbalancethe additional acid (H+) that is produced as a result of hydrolysis andthe PEG-albumin cross-linking, given the PEG concentrations required tomeet the therapeutic objectives for the composition. Thus, in thepreferred embodiment, a buffer material must be added to the albumin toprovide the required buffering capacity.

The buffer material must meet several criteria. The buffer material mustbe (1) non-toxic, (2) biocompatible, (3) possess a pK capable ofbuffering in the pH range where the desirable gelation time exists, and(4) must not interfere with the reaction of protein with the selectedPEG reactive ester. Amine-containing buffers do not meet criteria (4).

To meet criteria (3), the buffer material should have a bufferingcapacity at the desired cross-linking pH (i.e., according todicated byits pK) that is high enough to counterbalance the additional acid (H+)produced as a result of the cross-linking reaction and hydrolysis, i.e.,to keep the pH high enough to achieve the desired gelation time.

It has been discovered, through bench testing, that when cross-linkingthe SunBio PEG with albumin (Plasbumin), a range of gelation timesbetween an acceptable moderate time (about 30 seconds) to a rapid time(about 2 seconds) can be achieved by establishing a pH range from about8 (the moderate times) to about 10 (the rapid times). Ascertaining thecross-linking pH range aids in the selection of buffer materials havingpK's that can provide the requisite buffering capacity within the pHrange.

Phosphate, tris-hydroxymethylaminomethane (Tris), and carbonate are allnon-toxic, biocompatible buffers, thereby meeting criteria (1) and (2).Phosphate has a pK of about 7, which provides increased bufferingcapacity to albumin at pH's up to about 8.5. Tris has a pK of about 8,which provides increased buffering capacity to albumin at pH's up toabout 9.5. Addition of Tris to albumin (Plasbumin) at a concentration of60 mM approximately doubles the buffering capacity of the albumin at apH near 9. Carbonate has a pK of about 10, and provides increasedbuffering capacity to albumin in the higher pH ranges. Depending uponthe gellation time that is targeted, formulations of Tris, carbonate,and albumin can be used for the buffer material.

EXAMPLE Carbonate Buffer/Tris Buffer Formulations

Albumin (Human 25%, Plasbumin®-25 manufactured by Bayer Corporation) wasbuffered using Sodium Carbonate Anhydrous (Na₂CO₃) (FW 106.0)(“Carbonate Buffer”) mixed with Tris-hydroxymethylaminomethane(C₄H₁₁NO₃) (FW 121.1) (“Tris Buffer”). The buffered albumin formulations(2 cc) were mixed with 2 cc of the SunBio PEG (0.45 g of PEG suspendedin 2.2 cc of water), to provide 17% w/w PEG solids. The components weremixed in the manner described in Example 1. The pH of the bufferedalbumin formulation (albumin plus buffer material) and the gelation time(as described above) and were recorded.

Table 1 summarizes the results: TABLE 1 Albumin Carbonate Tris DeviceGelling (Human Buffer Buffer (Outside Time 25%) (ml) (grams) (grams) pHDiameter) (Seconds) 20 0 0.217 8.3 7 Fr 11 20 0 0.290 8.5 7 Fr 7-8 200.075 0.145 8.7 7 Fr 5-6 20 0.138 0.145 9.0 7 Fr 2-3

Table 1 shows rapid gelation times. This is believed due to the largerconcentration of multiple functionality PEG in the SunBio PEG, as wellas the enhanced buffering capacity that the Tris Buffer (pK 8) providesin the lower pH range (7 to 9). It is also believed that the gelationtime will also vary, given the same composition, according to the sizeand configuration of the delivery device. The addition of CarbonateBuffer (in the pH 8.7 and pH 9 compositions) leads to a further decreasein gelation time, at an increased pH.

Tests of pH 8.3 and pH 8.5 compositions in Table 1 have demonstratedthat both composition are successful in sealing femoral puncture sitesin sheep in 25 to 40 seconds. The tests also show that eithercomposition possesses sufficient cross-linking capacity to close aboutthe catheter tract following removal of the catheter upwards to twominutes after delivery of the material. Both compositions therebyreadily accommodate variations in procedure time.

Tests of pH 8.7 composition in Table 1 have also demonstrated that thecomposition is successful in sealing femoral puncture sites in sheep in25 to 40 seconds. The tests also show that, due to the more rapidgelation time, the composition does not possesses sufficientcross-linking capacity to consistently close about the catheter tractfollowing removal of the catheter two minutes after delivery of thematerial. In this respect, the pH 8.7 composition, despite its fastergelation time, is not as accommodating to changes in procedure time asthe pH 8.3 and pH 8.5 compositions, described above. For these reasons,the most preferred range for vessel puncture sealing is between pH 8.3and pH 8.5.

Further details of the material composition are found in copending U.S.patent application Ser. No. 09/780,014, filed Feb. 9, 2001, and entitled“Systems, Methods, and Compositions for Achieving Closure of VascularPuncture Sites.

REPRESENTATIVE EMBODIMENT

In a representative embodiment employed with a 7 FR device, the vial 94contains 600 mg±10% of lyophilized SunBio PEG-SG (4-arm polyethyleneglycol tetrasuccinimidyl glutarate—MW 10,500±1500). The lyophilizationprocess will be described in detail later. The syringe 98 contains 6 mlof water and 2 ml of buffered 25% w/w human serum albumin, USP. Thebuffered 25% albumin is made by adding 0.217 g. ofTris-hydroxymethlaminomethane (C₄H₁₁NO₃) (FW 121.1) (TRIS Buffer) to 20cc of Bayer Plasbumin®-25 to obtain a pH between 8.0 and 8.7, mostpreferably between 8.3 and 8.5. This composition is described in Table1.

The Lyophilization Process

The PEG material is moisture sensitive, i.e., it can be subject to rapiddegradation upon exposure to moisture. This moisture sensitivity canlimit the stability of the PEG material and thus its long-term storageor “shelf life.” Therefore, as previously noted, it may be desirablethat the PEG material be lyophilized and stored under vacuum or inertgas. During lyophilization, a solid substance is isolated from solutionby freezing the solution and evaporating the ice under vacuum. Theprocess removes essentially all moisture from the solid substance. Byremoving essentially all moisture from the PEG, the shelf life can besignificantly extended.

A representative lyophilization procedure employing a Stokes Lyophilizerfollows:

Sterilize Lyophilizer

In preparing lyophilizer, clean the chamber before use. Thesterilization cycle can be run, if needed, to clean the chamber. Beforesterilizing chamber, inspect for broken glass, stoppers, residualspilled product, and tape. Clean chamber shelves with alcohol. Check andreplace vacuum pump oil as required.

2. Pre-freeze lyophilizer at least 120 minutes before loading the vialsinto the lyophilizer.

3. Fill vials with 4 ml polymer solution (10% to 20% w/w solution, mostdesirably 15% w/w solution, PEG-SG in aqueous solution (sterile water)).

4. Place vials in trays into the lyophilizer.

5. Complete lyophilization according to the following table, to yield,in each vial 600 mg±10% of lyophilized PEG-SG material. Pre-FreezeSegment Segment Vacuum Ramp/ Number Description Temperature Time (mTorr)Soak 1 Loading −50° C.  1 minute Off Ramp 2 Pre-freeze −50° C. Max. timeOff Soak 3 Pre-freeze −50° C. 60 minutes Off Ramp 4 Pre-freeze −40° C.10 minutes 50 Ramp

Primary Dry Segment Segment Temper- Vacuum Ramp/ Number Descriptionature Time (mTorr) Soak 1 Primary +10° C. 1200 minutes 50 Ramp Drying 2Primary +20° C. 1200 minutes 50 Ramp Drying 3 Primary +20° C. 1920minutes 50 Soak Drying

Cycle End Segment Description Temperature Time Ramp/Soak N₂ Backfill+20° C. 30 min. to Soak 14.7 PSIA Stoppering +20° C. N/A N/A

In use, the 600 mg±10% lyophilized PEG-SG material in each vial isreconstituted with 2 ml buffered human serum albumin (25%) and 6 mlwater.

Representative Use of the System to Deliver Material Compositions toClose Vascular Puncture Sites

The Introduction Stage

(The Composition Liquid Phase)

In the first stage (see FIG. 19), the physician primes the selectedcatheter assembly 20 and selected introducer/mixing assembly 22 withsterile water or saline. The physician then introduces the selectedcatheter assembly 20 through the tissue track 34 partially into theblood vessel through the vascular puncture 36. As FIG. 19 shows, thestructure 64 is in a collapsed condition at this stage.

Typically, the catheter assembly 20 is introduced along a guide wire 32.As earlier explained and as shown in preceding FIGS. 17 and 18, theguide wire 32 will have been previously introduced percutaneously,through a wall of the blood vessel, to guide passage of a desiredtherapeutic or diagnostic instrument 30 into the blood vessel. As alsopreviously explained, the diameter of the outer catheter body 26 of thecatheter assembly 20 is preferably sized to seal, but not enlarge, thetissue track 34. In other words, the outside diameter of the outercatheter body 26 substantially matches the outside diameter of thevascular introducer 28 (by now retracted).

As FIG. 20 shows, the structure 64 is expanded within the blood vessel(as previously described). The physician applies back pressure on thecatheter assembly 20, bringing the expanded structure 64 into contactwith the interior of the vessel wall. By gauging the back pressure, thephysician locates the nozzles 60 outside the puncture site 36, as FIG.20 shows. The physician links the formative component assembly 18through the introducer/mixer assembly 22 to the catheter assembly 20 (asshown in FIG. 13).

Operation of the formative component assembly 18, as previouslydescribed, expresses the components 96 and 100, while in liquid form,through the mixer 88 and down the catheter assembly 20 toward thenozzles 60. The gelating components 138 flow out the nozzles 60 and intothe subcutaneous tissue surrounding the vessel, as FIG. 21 shows. Thecatheter assembly 20, which is sized to seal the tissue track 34, blockssubstantial flow in a path up the tissue track 34. Thus, the gelatingcomponents 138 are directed in a flow radially away from the axis of thecatheter assembly 20 and along the axis of the vessel, as FIG. 21 shows.

In FIG. 21, the nozzles 60 are arranged in a circumferentially spacedarray, as shown in FIG. 7A. The array is desirably close to the puncturesite 36. If the blood vessel has be accessed before in the same region,scar tissue may be present adjacent to the puncture site 36, and thenozzles 60, arranged as shown in FIG. 7A, may reside in the scar tissueregion. The scar tissue could interfere with the passage of the gelatingcomponents 138. In this circumstance, it may be desirable to arrange thenozzles 60 in the superior-inferior pattern shown in FIG. 7B, in whichanother array of superior nozzles 60B (located free of the scar tissueregion) are axially spaced away from the array of inferior nozzles 60A(located within the scar tissue region). In this arrangement, it isdesirable to size the superior nozzles 60B smaller than the inferiornozzles 60A. For example, the superior nozzles 60B can have an outsidediameter of about 0.020 inches, whereas the inferior nozzles 60A canhave an outside diameter of about 0.035 inches. The differential sizingof the nozzles 60A and 60B creates differential flow, creating apreferred normal flow path (of least flow resistance) through theinferior nozzles 60A, but allowing alternative flow through the superiornozzles 60B should increased flow resistance be encountered through theinferior nozzles 60A due to surrounding tissue morphology.

The spacing between the nozzles 60A and 60B can also vary. For example,the inferior nozzles 60A can be spaced from the structure 64 by 3 to 10mm, whereas the superior nozzles 60B can be further spaced 5 to 15 mmfrom the structure 64.

The size of the catheter assembly 20 is selected according to theoutside diameter of the introducer sheath 28 used during the precedingtherapeutic or diagnostic procedure, during which the arteriotomy wasmade. For example, a 6 Fr introducer sheath 28 typically has an outsidediameter of 7 Fr, so a 7 Fr diameter catheter assembly 20 is selected toseal the arteriotomy after removal of the introducer sheath 28. Thegelating composition 138 is delivered in a liquid state adjacent to thearteriotomy, while the catheter assembly 20 prevents the liquid fromfilling the tissue track 34. This feature ensures that the materialcomposition remains at the arteriotomy for maximum efficacy.

The incoming flow, directed in this manner, creates a tissue space aboutthe puncture site 36 along the axis of the vessel. The gelatingcomponents 138 fill this space.

In the gelation process, the electrophilic component and thenucleophilic component cross-link, and the developing composition 138gains cohesive strength to close the puncture site 36. The electrophiliccomponent also begins to cross-link with nucleophilic groups on thesurrounding tissue mass. Adhesive strength forms, which begins to adherethe developing composition to the surrounding tissue mass.

During the introduction stage, before internal cohesive and tissueadhesive strengths fully develop, a portion of the gelating components138 can enter the blood vessel through the puncture site 36. Uponentering the blood stream, the gelating components 138 will immediatelyexperience physical dilution. The dilution expands the distance betweenthe electrophilic component and the nucleophilic component, makingcross-linking difficult. In addition, the diluted components nowexperience an environment having a pH (7.3 to 7.4) lower than the aneffective reactive pH for cross-linking (which is above 8) (as anexample, a typical gelation time at pH 8.3 is about 15 to 20 seconds,whereas a typical gelation time at pH 7.4 is over 10 minutes). As aresult, incidence of cross-linking within the blood vessel, to form thehydrogel composition, is only a fraction of what it is outside thevessel, where gelation continues.

Furthermore, the diluted electrophilic component will absorbnucleophilic proteins present in the blood. This reaction furtherreduces the reactivity of the electrophilic component. In blood, thediluted electrophilic component is transformed into a biocompatible,non-reactive entity, which can be readily cleared by the kidneys andexcreted. The diluted nucleophilic component 100 is a naturallyoccurring protein that is handled in normal ways by the body.

The Introduction Stage (The Composition Liquid Phase) preferably lastabout 5 to 30 seconds from the time the physician begins to mix thecomponents 96 and 100.

The Localized Compression Stage (The Semi-Solid Composition Phase)

The second stage begins after the physician has delivered the entireprescribed volume of components 96 and 100 to the tissue mass of thevessel puncture site 36 and allowed the cross-linking of the components96 and 100 to progress to the point where a semi-solid gel occupies theformed tissue space.

At this point (as FIG. 22 shows), the physician collapses the structure64 and withdraws the catheter assembly 20 and guide wire 32 from thetissue track 34. The physician now simultaneously applies localized andtemporary compression to the exterior skin surface surrounding thetissue track 34.

The application of localized pressure serves two purposes. It is not toprevent blood flow through the tissue track 34, as cross-linking of thecomponents 96 and 100 has already proceeded to create a semi-solid gelhaving sufficient cohesive and adhesive strength to impede blood flowfrom the puncture site. Rather, the localized pressure serves tocompress the tissue mass about the semi-solid gel mass. This compressionbrings the semi-solid gel mass into intimate contact with surroundingtissue mass, while the final stages of cross-linking and gelation takeplace.

Under localized compression pressure, any remnant catheter trackexisting through the gel mass will also be closed.

Under localized compression pressure, surface contact between theadhesive gel mass and tissue is also increased, to promote thecross-linking reaction with nucleophilic groups in the surroundingtissue mass. Adhesive strength between the gel mass and tissue isthereby allowed to fully develop, to firmly adhere the gel mass to thesurrounding tissue as the solid composition 136 forms in situ.

During this stage, blood will also contact the vessel-side, exposedportion of the gel mass, which now covers the tissue puncture site. Theelectrophilic component will absorb nucleophilic proteins present in theblood, forming a biocompatible surface on the inside of the vessel.

The Localized Compression Stage (The Composition Semi-Solid (Gel) Phase)preferably last about 3 to 10 minutes from the time the physicianwithdraws the catheter assembly 20.

The Hemostasis Stage The Composition Solid Stage

At the end of the Localized Compression Stage, the solid composition 136has formed (as FIG. 22 shows). Hemostasis has been achieved. Theindividual is free to ambulate and quickly return to normal day-to-dayfunctions.

The mechanical properties of the solid composition 136 are such to forma mechanical barrier. The composition 136 is well tolerated by the body,without invoking a severe foreign body response.

The mechanical properties of the hydrogel are controlled, in part, bythe number of crosslinks in the hydrogel network as well as the distancebetween crosslinks. Both the number of crosslinks and the distancebetween crosslinks are dependent on the functionality, concentration,and molecular weight of the polymer and the protein.

Functionality, or the number of reactive groups per molecule, affectsthe mechanical properties of the resulting hydrogel by influencing boththe number of and distance between crosslinks. As discussed previously,the utility of a given polymer significantly increases when thefunctionality is increased to be greater than or equal to three. Theobserved incremental increase in functionality occurs when thefunctionality is increased from two to three, and again when thefunctionality is increased from three to four. By increasing thefunctionality of the polymer or protein at a constant concentration, theconcentration of crosslinking groups available for reaction areincreased and more crosslinks are formed. However, increased mechanicalproperties cannot be controlled with functionality alone. Ultimately,the steric hindrances of the protein or polymer to which the reactivegroups are attached predominate and further changes in the mechanicalproperties of the hydrogel are not observed. The effect of functionalityis saturated when the functionality reaches about four.

The concentration of the protein and polymer also affect the mechanicalproperties of the resulting hydrogel by influencing both the number ofand distance between crosslinks. Increasing the protein and polymerconcentration increases the number of available crosslinking groups,thereby increasing the strength of the hydrogel. However, decreases inthe elasticity of the hydrogel are observed as the concentration of theprotein and polymer is increased. The effects on the mechanicalproperties by concentration are limited by the solubility of the proteinand polymer.

The polymer and protein molecular weight affects the mechanicalproperties of the resulting hydrogel by influencing both the number ofand distance between crosslinks. Increasing the molecular weight of theprotein and polymer decreases the number of available crosslinkinggroups, thereby decreasing the strength of the hydrogel. However,increases in the elasticity of the hydrogel are observed with increasingmolecular weight of the protein and polymer. Low molecular weightproteins and polymers result in hydrogels that are strong, but brittle.Higher molecular weight proteins and polymers result in weaker, but moreelastic gels. The effects on the mechanical properties by molecularweight are limited by the solubility of the protein and polymer.However, consideration to the ability of the body to eliminate thepolymer should be made, as large molecular weight polymers are difficultto clear.

The Degradation Stage (The Composition Re-Absorption Phase)

Over a controlled period, the material composition is degraded byphysiological mechanisms. Histological studies have shown a foreign bodyresponse consistent with a biodegradable material, such as VICRYL™sutures. As the material is degraded, the tissue returns to a quiescentstate. The molecules of the degraded genus hydrogel composition arecleared from the bloodstream by the kidneys and eliminated from the bodyin the urine. In a preferred embodiment of the invention, the materialloses its physical strength during the first fifteen days, and totallyresorbs in about four to eight weeks, depending upon the person's bodymass.

The foregoing is considered as illustrative only of the principles ofthe invention. Furthermore, since numerous modifications and changeswill readily occur to those skilled in the art, it is not desired tolimit the invention to the exact construction and operation shown anddescribed. While the preferred embodiment has been described, thedetails may be changed without departing from the invention, which isdefined by the claims.

1. A method comprising providing a first container holding abiocompatible polymer in lyophilized form, the biocompatible polymerhaving a functionality equal to or greater than three, providing asecond container holding a protein solution introducing the proteinsolution into the first container for mixing with the polymer inlyophilized form to reconstitute the polymer and form a mixture,wherein, upon mixing, the protein solution and the polymer cross-link.2. A method according to claim 1 wherein the biocompatible polymercomprises poly(ethylene glycol) PEG.
 3. A method according to claim 1wherein the protein solution comprises albumin.
 4. A system comprising afirst container holding a biocompatible polymer in lyophilized form, thebiocompatible polymer having a functionality equal to or greater thanthree, a second container holding a protein solution an applicator tointroduce the protein solution into the first container for mixing withthe polymer in lyophilized form to reconstitute the polymer and form amixture, wherein, upon mixing, the protein solution and the polymercross-link.
 5. A system according to claim 4 wherein the biocompatiblepolymer comprises poly(ethylene glycol) PEG.
 6. A system according toclaim 4 wherein the protein solution comprises albumin.
 7. A method oftreating tissue comprising lyophilizing a biocompatible polymer having afunctionality equal to or greater than three; providing a proteinsolution, mixing the protein solution with the lyophilized polymer toreconstitute the polymer and form a mixture, wherein, upon mixing, theprotein solution and the polymer cross-link to form a materialcomposition, and applying the material composition to a tissue region.8. A method according to claim 7 wherein the biocompatible polymercomprises poly(ethylene glycol) PEG.
 9. A method according to claim 7wherein the protein solution comprises albumin.
 10. A method comprisinglyophilizing a biocompatible polymer having a functionality equal to orgreater than three, providing a protein solution, and mixing the proteinsolution with the lyophilized polymer to reconstitute the polymer andform a mixture, wherein, upon mixing, the protein solution and thepolymer cross-link to form a material composition.
 11. A methodaccording to claim 10 wherein the biocompatible polymer comprisespoly(ethylene glycol) PEG.
 12. A method according to claim 10 whereinthe protein solution comprises albumin.
 13. A method of treating tissuecomprising lyophilizing a biocompatible polymer having a functionalityequal to or greater than three, providing a protein solution, providinga catheter, mixing the protein solution with the lyophilized polymer toreconstitute the polymer and form a mixture, wherein, upon mixing, theprotein solution and the polymer cross-link to form a materialcomposition, and applying the material composition through the catheterto a tissue region.
 14. A method according to claim 13 wherein thebiocompatible polymer comprises poly(ethylene glycol) PEG.
 15. A methodaccording to claim 13 wherein the protein solution comprises albumin.